Abstract
Background/Aim: Rhenium(I)-diselenoether (Re-diSe) is a promising anticancer agent composed of one rhenium and two selenium atoms. Its effectiveness was established in inhibiting cancer cells while maintaining low toxicity toward normal cells at a 5 μM dose for 120 hours in MDA-MB-231 cells. In MDA-MB-231 breast tumor-bearing mice, anti-tumor and anti-metastatic effects were observed at a 10 mg/kg dose. However, contradictory results were observed in the 4T1 breast cancer model, where a dose of 60 mg/kg had a pro-tumor effect. To address these discrepancies, the efficacy of Re-diSe at the effective 10 mg/kg dose was validated in a transplanted MDA-MB-231 breast tumor model using the chicken chorioallantoic membrane assay. Materials and Methods: MDA-MB-231 cancer cells were xenografted onto the chicken chorioallantoic membrane (CAM), and daily drug administration was carried out for nine days at doses of 0.1, 1, and 10 mg/kg. At the study’s conclusion, a standard histological analysis was conducted. Results: The low dose of 0.1 mg/kg showed a significant reduction in tumor weights compared to controls. The 1 mg/kg dose resulted in an increased inflammation score but did not induce a significant difference in tumor weights compared to the 0.1 mg/kg dose. Notably, at the 10 mg/kg dose, six out of 11 treated embryos displayed no visible signs of tumors. These tumors exhibited extensive tumor necrosis and significant infiltration by inflammatory cells. Conclusion: In this particular model, the anticancer efficacy of Re-diSe was achieved at the low dose of 0.1 mg/kg. The higher dose of 10 mg/kg, while eliminating visible tumors, might have immune-mediated effects, as indicated by substantial tumor necrosis and infiltration by inflammatory cells. Overall, this study successfully demonstrated the effectiveness of Re-diSe as an anticancer agent.
The rhenium(I)-diselenoether (Re-diSe) is a metal-based drug that displays a fac-[Re(CO)3]-core tightly bonded to 3,7-diselenanonanedioic acid disodium salt ligand stabilizing the metal and allowing water solubility (1). It was found to selectively inhibit the growth of cancer cells versus normal cells, decreasing selectively the production of reactive oxygen species (ROS), transforming growth factor-beta (TGF-β), vascular epidermal growth factor A (VEGF-A) and insulin growth factor 1 (IGF1) by the cancer cells (2), even when the culture medium of the cells was enriched in inflammatory cytokines (3). Its antitumor activity was demonstrated in an experimental model of triple-negative breast cancer (TNBC) with MDA-MB-231 orthotopically transplanted tumor-bearing nude mice, after an oral administration of 10 mg/kg for 28 days, both on the primary tumors and the metastases (4). A significant antitumor effect was confirmed in the same model after a daily intraperitoneal administration for 28 days, without differences between the doses of 10 and 40 mg/kg, while higher doses induced a toxicity without improving efficiency (5). Further experimental studies showed controversial results. A pro-tumor effect was observed at daily oral doses of 5 or 10 mg/kg Re-diSe for six weeks in the MDA-MB-231 model, but in immunodeficient mice treated with a total-body irradiation prior to the transplantation of the tumor cells, suppressing thus their immune defense. High daily oral doses of 60 mg/kg Re-diSe induced a pro-tumor effect in 4T1- transplanted tumors in non-immune deficient mice, while no effect on the tumor growth was noted at lower doses of 0.1, 1, and 10 mg/kg. It was concluded that the choice of the model was crucial to determine the efficacy of Re-diSe. Therefore, we wanted to confirm the efficacy of the Re-diSe drug in MDA-MB-231 tumor-bearing mice at the dose of 10 mg/kg, which already showed an anticancer activity in this model of triple-negative breast cancer, and at lower doses.
The chicken chorioallantoic membrane assay has the advantage to rapidly develop within 8 days measurable tumors and represents an alternative to experiments in tumor-transplanted nude mice (6-8). We used MDA-MB-231 breast cancer cells xenografted on the chicken chorioallantoic membrane (CAM) to assess the efficacy of the Re-diSe drug. To evaluate a dose-dependent effect, Re-diSe was administered for nine days at three different doses: 0.1, 1, and 10 mg/kg from the embryonic developmental day 10 (EDD10) to EDD18; the embryos were sacrificed at EDD19. The efficacy was evaluated through the measurement of the tumor weights and by a standard histological analysis performed on fixed tumors at the end of the experiment. The hematoxylin and eosin histological analysis allowed to study the tumor cells, the infiltration by inflammatory cells, and the tumor necrosis. The toxicity was evaluated by comparing the number of dead embryos in Re-diSe-treated and control (vehicle) embryos.
Materials and Methods
The chicken embryo CAM assay was performed by INOVOTION SAS (La Tronche, France). No ethical approval is required for scientific experimentation using oviparous embryos (decree number 2013-118, February 1, 2013; art. R214-88) according to French legislation.
Synthesis. Rhenium(I)diseleno-ether (Re-diSe) was synthesized by ligand exchange from pentacarbonylchlororhenium with 3,7-diselenanonanedioic acid. The obtained dicarboxylic acid rhenium complex was converted into its water soluble bis sodium salt upon treatment with sodium carbonate as reported earlier (1).
Design. Fertilized white Leghorn eggs (Couvoir Hubert, Guilberville, France) were incubated at 37.5°C with 50% relative humidity for nine days. At EDD9, eggs were randomized into groups and the CAM was dropped down by drilling a small hole through the eggshell into the air sac, and a 1 cm2 window was cut in the eggshell above the CAM. Then, 1×106 MDA-MB-231 cells (from ATCC, Manassas, VA, USA) in 50 μl of culture media were inoculated onto the egg’s CAM.
Treatments. From EDD10 to EDD18, eggs received either vehicle (water, n=16), Re-diSe [1] (0.1 mg/kg, n=17), Re-diSe [2] (1 mg/kg, n=17) and Re-diSe [3] (10 mg/kg, n=19) every day.
Efficacy. Evaluation of the efficacy on the primary tumors: at EDD19, tumors which were attached on the upper portion of the CAM were removed, washed with PBS buffer, and then fixed in 4% PFA for 48 h. Tumors were carefully cut away from normal CAM tissue and weighed.
Histological observations. PFA-fixed tumors were trimmed and embedded in paraffin cassettes (n=11 per group). Paraffin sections were cut (4 μm thick) and put on glass slides. Slides were stained with hematoxylin & eosin for general histopathological evaluation.
Non-overlapping fields were graded by a semi-quantitative scoring system for the presence of pathological changes (n=10 fields per tumor).
Pictures were taken using a microscope (Olympus BX60, serial NO.7D04032, Rungis, France) at objective magnification X1.25 and X10, and microscope’s camera (Olympus DP73, serial NO. 0H05504).
It has been demonstrated that the tumor inflammatory cell infiltrate and the tumor necrosis could be assessed on hematoxylin and eosin-stained sections (9-11). The intensity of overall inflammatory cell reaction, numbers of neutrophilic and eosinophilic granulocytes, lymphoid cells and macrophages is scored using this method (12).
Inflammation was scored from 0 to 3 with 0: scant or absent stroma cells, 1: stroma cells obviously present but markedly less than tumor cells, 2: stroma cells roughly equal to tumor cells and 3: predominantly stroma cells.
Tumor necrosis was scored from 0 to 5 with 0: no necrosis, 1: small foci of necrosis, 2: necrosis in <50% of the field, 3: necrosis in 50-75% of the field, 4: necrosis in 75-90% of the field and 5: necrosis in >90% of the field.
Statistical analysis. A one-way ANOVA analysis with Tukey’s multiple comparison post-test was performed on the dataset. For all analyses, statistically significant differences between groups are indicated on the graphs using stars as follows: - No star: No statistically significantly different (p-value >0.05); - One star (*): 0.05 ≥ p-value >0.01; - Two stars (**): 0.01 ≥ p-value >0.001; - Three stars (***): 0.001 ≥ p-value ≥0.0001; - Four stars (****): p-value ≤0.0001.
Results
Efficacy. A significant decrease of the tumor weights was observed at the dose of 0.1 mg/kg Re-diSe versus controls, from 25.74±1.95 mg in controls to 16.73±1.98 mg. The tumor weights in the group treated with 1 mg/kg (15.38±1.92 mg) did not significantly differ compared to those with the 0.1 mg/kg doses. No visible tumors were observed in 6/11 embryos treated with 10 mg/kg. In the other 5/11 embryos treated with this dose, the tumor weights significantly decreased to 8.74±1.81 mg by comparison with controls (Figure 1). The remarkable visual difference of the CAM between controls and the group treated at the dose of 10 mg/kg is shown in Figure 2. Table I emphasizes the absence of visible tumor in embryos treated with 10 mg/kg.
Average tumor weights (±SEM; n=11-14 embryos per group and n=5 in Re-diSe [3]). Human MDA-MB-231 breast cancer cells were grafted onto the chorioallantoic membrane and treated daily with vehicle (Neg. Ctrl.), Re-diSe at 0.1 mg/kg (Re-diSe [1]), 1 mg/kg (Re-diSe [2]) and 10 mg/kg (Re-diSe [3]). Tumors were collected 10 days after the graft, fixed in PFA and weighed. Only 5/11 tumors were visible in Re-diSe [3] and therefore included in the statistical analysis and shown in this figure. (**): 0.01 ≥ p-value >0.001, (***): 0.001 ≥ p-value ≥0.0001.
Illustrative pictures of the typical aspect of an MDA-MB-231 tumor grown on the CAM in the Neg. Ctrl. group (A) and the inflammatory membrane without visible tumor in 6/11 Re-diSe [3] treated embryos (B).
Absence of visible tumor in 6/11 Re-diSe [3] treated embryos.
Toxicity. There is a known lethal effect of the transplantation of the tumor cells on the embryos. The number of deaths was increased in all treated groups versus controls, but not significantly. The results are shown in Figure 3.
Kaplan–Meier curve summarizing the embryo survival of Re-diSe-treated eggs. Sixteen to nineteen embryos per group were grafted with MDA-MB-231 cancer cells at developmental day 9 (EDD9). Viability of the embryos was examined daily, and the death index recorded up to EDD19. Log-rank (Mantel–Cox) statistical test was used to analyze the results. p-Values are shown on the right part of the graph.
Histological evaluation. In controls, tumors consisted of a central, localized, nodular dense mass with round to spindle shape, large neoplastic cells with a very high mitotic activity and with clear cell borders. The central core was surrounded by a very loose network of neoplastic cells. High grade of nuclear pleomorphism and many multi-nucleated giant cells were observed in the neoplastic cells population. The score of inflammation cells was mild.
In the group treated with 10 mg/kg Re-diSe, 6 of the 11 samples showed only embryo membranes with very few surviving neoplastic cells and some necrotic material. The physiological aspect of the CAM was altered in these samples (thickening, absence of vascularization and whitening of the membrane at the grafting site).
The score of inflammation did not vary between controls (1.455±0.157) and embryos treated with 0.1 mg/kg Re-diSe (1.545±0.157), but significantly increased upon treatment with 1 mg/kg Re-diSe (2.091±0.162), and 10 mg/kg Re-diSe (2.091±0.090).
The score of tumor necrosis increased from 2±0.233 in controls to 2.182±0.181 at the dose of 0.1 mg/kg (N.S), and to 2.636±0.203 at the dose of 1 mg/kg (N.S). The tumor necrosis was highly significantly increased in the group treated with 10 mg/kg (4.182±0.325) and significantly compared with the two other treated groups. The results are shown in Figure 4.
Histological analysis of tumors treated with Re-diSe. Tumors fixed in PFA4% were included in paraffin blocks, sliced, and stained with hematoxylin and eosin. The average score (±SEM) for inflammation (A) and necrosis (B) was assessed by a histopathologist using a semi-quantitative scoring technique (see Materials and Methods). Ordinary one-way ANOVA with Tukey’s multiple comparison post-test was used to analyze the results. n=11 tumors per group. (*): 0.05 ≥ p-value >0.01; (***): 0.001 ≥ p-value ≥ 0.0001; (****): p-value ≤0.0001.
Microscopical pictures. Images of the microscopic observations in Figure 5 illustrate the remarkable rarefaction of the tumors cells, the infiltration by inflammatory cells and the huge necrosis of the tumors at the dose of 10 mg/kg with intermediate results at the lower doses. The CAM was altered at the dose of 10 mg/kg and appeared inflammatory.
Illustrative pictures of hematoxylin and eosin stained tumors. Tumors fixed in PFA4% were included in paraffin blocks, trimmed, and stained with hematoxylin and eosin. One representative image is provided for each group at two magnifications. A and B) Tumor morphology in the control group; C and D) 0.1 mg/kg Re-diSe; E and F) 1 mg/kg Re-diSe; G and H: 10 mg/kg Re-diSe. Pink staining (eosin) is specific for cell’s cytoplasm and extracellular matrix, while purple staining (hematoxylin) is specific for cell’s nucleus. Yellow arrows indicate necrotic areas and red arrows proliferative ones. Scale bar=500 μm (A, C, E & G) and 50 μm (B, D, F & H).
Discussion
Efficacy. The efficacy of Re-diSe was demonstrated at a dose as low as 0.1 mg/kg for a relatively short duration of administration, with a significant decrease in the tumor weight compared with the controls. Increasing the dose to 1 mg/kg did not decrease the tumor weight but induced an increase in the number of inflammatory cells. At the dose of 10 mg/kg, the decrease of the tumor weights was highly significant compared with the controls. This was confirmed by the histological analysis, which showed a rarefaction of the tumor cells or even a tumor necrosis and an increase in the number of inflammatory cells. The tumor weights did not significantly vary between 1 and 10 mg/kg but the 6/11 embryos without visible tumors were excluded from the statistics. Only measurable tumors in 5/11 embryos were included in the statistical analysis. In any case, at the dose of 10 mg/kg a remarkable anticancer activity was achieved.
Toxicity. There was an increase in the number of deaths of embryos in the treated groups, but the difference was not significant versus controls. The toxicity of Re-diSe was previously evaluated in other in vivo models. The toxicity observed in mice varied according to the mode of administration. A toxicity was noted after intraperitoneal (IP) injections (5). The 60 mg/kg dose was considered as the maximum tolerated dose (MTD) for a four-week daily IP administration, 75 mg/kg was the 50% lethal dose (LD50) dose and 40 mg/kg the LD10. Finally, the 10 mg/kg-treated group showed no sign of toxicity, and this was the no-adverse toxicity level. However, the oral administration of Re-diSe did not induce any sign of toxicity at the daily dose of 10 mg/kg for six weeks, even in combination with a weekly administration of paclitaxel during the last two weeks of treatment (13). Similar results were obtained with the daily dose of 60 mg/kg for 3 weeks (14). The mode of administration in the chicken embryo model is similar to the IP injection as the cells are directly inoculated on the membrane. An oral administration should be less toxic than a parenteral route, with the advantage to allow repeated administrations. The bioavailability of Re-diSe after an oral administration was demonstrated by the high and dose-dependent plasma concentrations of Re (15) as well as the dose-dependent tissue and tumor Re uptake (13, 16).
Inflammation/Immune cell infiltration. We observed a significant increase in the number of inflammatory cells in tumors treated with 1 and 10 mg/kg, but not in the group treated with 0.1 mg/kg, which induced a significant reduction of the tumor weights.
The term of inflammation is usual in histopathology, but microscopic observations without immuno-histochemistry assays cannot distinguish immune cells with pro-inflammatory properties from immune cells with anti-inflammatory properties. Moreover, cells of the tumor microenvironment (TME) are multiple. Lymphocytes, macrophages and dendritic cells are the main immune cells of the TME but tumor-associated stromal cells (TASCs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs), tumor-associated endothelial cells (TECs), mesenchymal stromal cells (MSCs) and cancer-associated adipocytes (CAA) also participate to the cancer development (17-19). There is a great heterogeneity between all these cells. In order to understand the immune effects of Re-diSe, we first give an overview on immune suppressive or immune stimulatory cells.
Immune suppressive and immune stimulatory cells. Myeloid cells, comprised macrophages, dendritic cells, monocytes, and granulocytes, represent a major component of the TME with either immune suppressive or immune stimulatory roles (20). They also play an important role in phagocytosis and antigen presentation to T-cells (21).
In the pathological state of activation, mainly induced by a lipid peroxidation, myeloid cells have an immune-suppressive role by inhibiting T cell immunity and the cytolytic function of intra-tumoral natural killer (NK) cells through the production of IL-10 and TGF-β and are defined as “myeloid-derived suppressor cells” (MDSCs) (22, 23). The granulocytic myeloid-derived suppressor cell (G-MDSC) is the most widely distributed subtype in tumors (24).
Tumor-associated macrophages (TAMs) may have pro- or anti-inflammatory properties (25) and their role is primordial in the cancer development (26). The significant incidence of macrophage infiltration is commonly correlated with an unfavorable prognosis, but it could be more relevant to characterize the type of macrophages.
While M2 macrophages exert an immune-suppressive effect, M1 macrophages stimulate inflammation against cancer cells (27). The therapeutic strategy could be to increase the number of M1-like macrophages and to decrease the M2-like macrophages but selectively in the tumor to avoid a dangerous systemic inflammation by M1-like macrophages or an excessive lack of function of M2-like macrophages.
T lymphocytes are the principal component of tumor-infiltrating lymphocytes (TILs). The number of TILs depends on the subtype of cancer (28-30). In breast cancers, the highest level of TILs was observed in triple-negative breast cancer (TNBC) which is the most aggressive subtype (31). However, the role of TILs may differ according to the type of lymphocytes present in the TME. The distinction between the involved lymphocytes could be more important than measuring their total number to evaluate their effects in cancer progression and invasiveness. CD8+ cytotoxic T cells mediate effective anti- tumor immunity and are the most powerful effectors in the anticancer immune response (32). CD4+ helper T cells (Th) may have distinct roles according to their subtypes, either Th1 or Th2 (33, 34). The differentiation of T-helper cells to Th1 and Th2 subsets is of prognostic value (35). CD25+CD4+ regulatory T cells (Tregs) are highly immune-suppressive, expressing FoxP3 (36). The highest levels of FOXP3+ cells are observed in TNBC (28). NK cells exert cytotoxic functions, eradicating tumor cells through cytolytic granules and cooperating with other innate and adaptive immune cells through proinflammatory cytokines (37).
Anti-inflammatory and pro-tumor cells are M2-type macrophages, regulatory T (Treg) cells, polymorphonuclear (PMN) and monocytic (M) MDSCs. They are considered immune-suppressive cells. They have been shown to be significantly increased in the tumor immune microenvironment (TIME (17). Th2 cells have also been linked to tumor promotion and positively correlated with Treg cells (38).
Pro-Inflammatory and anti-tumor cells are M1-type macrophages, CD8+ cytotoxic T cells and Th1 cells. They mediate an effective anti- tumor immunity in advanced cancers and facilitate the recruitment and activation of NK cells against cancer (39).
Cysteine proteases-mediated immune effects of Re-diSe on the polarization of macrophages. It was demonstrated that Re-diSe significantly increased the number of M1-like macrophages and significantly decreased the number of M2-like macrophages (40). To induce the polarization into M1 macrophages, murine Raw 264 or human THP-1 macrophages were previously treated with liposaccharides (LPS), while to induce the polarization into M2, the cells were stimulated with interleukin IL-6. The stimulation with LPS reflects the increased inflammatory situation observed in the TME. The increased number of M1 macrophages induced by Re-diSe could explain the destruction of the tumor cells observed in our study, while the decreased number of M2 macrophages could have counteracted the immune-resistance of the tumor cells. It is also important to note that Re-diSe had no effect on non-prior stimulated macrophages, thus in the absence of inflammation.
The effects of Re-diSe on the polarization of the macrophages were explained by the effects of Re-diSe on the cysteine proteases cathepsins B and S (20). It was demonstrated that Re-diSe decreased the production of cathepsins B and S in macrophages (40) and in cancer cells (41). Cathepsins B and S are over-expressed in cancer cells and in macrophages (42). They are involved in cancer progression and in the immune/inflammation response of the cells of the TME (43). Serum levels of cathepsin S increased with the stage of cancer and are over-expressed at late metastatic stage (44). Inhibition of the activity of cathepsins B and S induced antitumor effects (45-49), and a shift from M2 to M1-like phenotype, with an increased expression of autophagy- and lysosome-associated marker genes, changes in lysosomal activity, fatty acid metabolism, synthesis of pro-inflammatory mediators and reduced adenosine triphosphate (ATP) levels (50).
Re-diSe could have an immunotherapeutic activity in addition to its selective inhibitory effects on cancer cells when cysteine proteases are produced in excess. However, cysteine proteases are necessary for an optimal function of immune cells. The objective should be to normalize the production of cysteine proteases when they are over-expressed and not to decrease them below the optimal level required for a normal function of the immune cells.
Reactive oxygen species (ROS)/Reactive nitrogen species (RNS) mediated-effects of Re-diSe on immune and cancer cells. The binding of the Re atom of the Re-diSe drug with thiol (SH) groups was clearly demonstrated in the cases of N-acetylcysteine and of glutathione (GSH) (40). By this mechanism, Re-diSe will affect the redox status of immune and cancer cells.
Binding of Re to SH groups. The SH groups of redox-active Cys residues have high nucleophilic properties, a high affinity metal binding and the ability to form disulfide bonds with an exchange between reduced dithiol-containing molecules and oxidized disulfide-containing molecules (51). Protein and non-protein thiols are the first targets of ROS (52). Through the binding of Re-diSe with SH groups, the Re-diSe could affect protein oxidation, which commonly occurs at thiol-containing cysteine residues, by preventing disulfide bridge formation (52). There are many SH-rich proteins in cancer cells. For example, Park et al. identified 194 reactive SH-containing proteins in prostate cancer cells (53). It was also observed that cationic Re complexes could covalently bind to SH-rich mitochondrial components (54). The redox status of the immune and cancer cells may thus be modified by the Re-diSe treatment. In cancer cells, there is usually a pro-oxidative status (55). Oxidation-reduction in cancer cells are in favor of cellular oxidation with a more pronounced pro-oxidant status when the stage of the cancer is more advanced (56). MDSCs are also induced by oxidation, as previously described. It is via an oxidative-stress-associated mechanism that apoptotic Treg cells achieved superior suppressor function, inhibiting spontaneous and checkpoint- blockade-induced antitumor immunity (57).
The particular case of GSH. The binding of the Re atom of Re-diSe with GSH may have major consequences. The GSH levels are high in cancer cells to protect them from the oxidative damage induced by anticancer drugs (58). Excess GSH promotes tumor progression, where elevated levels correlate with increased metastasis (59). Depletion of GSH has been proposed as a new strategy against cancer, with ferroptosis as a possible result (60). Ferroptosis could be a mode of Re-diSe-induced death at high doses, resulting in significant Cys depletion (61). At lower doses reaching and respecting the normal values of thiols, Re-diSe could induce favorable immune effects on the polarization of macrophages.
Decreased production of ROS, TGF-β1, VEGF-A, and IGF-1. A dose-dependent decrease in the production of ROS, TGF-β1, VEGF-A, and IGF-1 was observed in MDA-MB-231 breast cancer cells treated with Re-diSe, but not in normal HEK-293 cells (2). The Re-diSe complex was more effective than its di-Se ligand, suggesting that Re plays a critical role in the anticancer activity.
ROS play an important role as signaling messengers in the immune system. They regulate T cell immune response in the TME (62), but also the fate and function of MDSCs (63). Tumor growth is associated with the accumulation of immature myeloid cells (ImC) with significantly higher levels of ROS than ImC isolated from tumor-free animals. They suppress Ag-specific CD8+ T cells via direct cell-cell contact. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells (64). The decreased ROS production by Re-diSe could thus have indirect effects on innate and adaptive immunity.
TGF-β1, VEGF-A, and IGF-1 are redox-dependent factors, greatly involved in cancer progression and immune-suppression. TGF-β regulates infiltration of inflammatory/immune cells and cancer-associated fibroblasts in the TME causing direct changes in tumor cells. Neutralizing TGF-β enhanced CD8+ T-cell- and NK-cell-mediated anti-tumor immune responses (65). A decrease of VEGF increased NK cell mediated lysis of an ovarian cancer cell line and enhanced the cytolytic activity of immune T cells, without toxicity for the immune cells at doses providing cytotoxic effects for the cancer cells (66). Insulin/IGF-1 signaling promotes immunosuppression via the STAT3 pathway (67) and stimulates the expansion of Tregs, increasing their immunosuppressive activity (68).
Through its action on ROS, TGF-β1, VEGF-A, and IGF-1 the Re-diSe could then increase the number of cytotoxic T cells and NK cells, while decreasing MDSCs cells, and the expansion of Tregs, fighting the immune-resistance of cancer cells.
Binding of Re with DNA. The binding of Re to guanine bases was demonstrated via the formation of mono- or bis-adducts with Re-diSe (4). Re was found in the nucleus of cancer cells exposed to Re-diSe (16). Nearly all tricarbonyl(I)-Re complexes form reversible bonds with either adenine or guanine bases, in contrast to the binding of cisplatin with guanine bases which is irreversible (1). However, the mitochondrial DNA could also be a target of Re-diSe. For example, it was shown that the binding of a tricarbonyl(I)-Re complex with doxorubicin diverted the normal accumulation of doxorubicin from the nucleus to mitochondria (69). Moreover, it has been recently shown that mitochondria were a target of third row transition metal-based anticancer complexes (70). The mitochondrial DNA is greatly involved in ROS and RNS productions and the binding of Re with the mitochondrial DNA could be related to its effect on reactive species. Further studies are necessary to confirm this hypothesis.
Tumor necrosis. The role of inflammatory/immune cells in the necrosis of the tumor is highly suggested by the results observed at the dose of 10 mg/kg, with increased inflammation. It is the first time that the efficacy of Re-diSe is correlated with immune cells infiltration with a remarkable tumor necrosis at the dose of 10 mg/kg, which is the main result of this study.
An increased number of M1-like macrophages could be expected as already emphasized, with a decrease in M2-like macrophages. However, via the effects of Re-diSe on multiple pathways, an activation of NK cells and an increased number of cytotoxic cells, combined with a decreased role of MDSCs and Tregs are expected. Re-diSe could then be considered a new immunotherapeutic drug.
In any case, the doses of Re-diSe should be managed to reach and respect the normal levels of GSH, cysteine proteases, ROS and RNS in the immune cells, while inhibiting the growth of cancer cells.
An autophagic type of death of cancer cells was observed with other Re compounds and linked to lysosomal dysfunctions involving cathepsin protease B (71). A paraptosis pathway for cell killing with vacuole formations was also observed in prostate cancer PC3 cells exposed to Re complexes (72).
The mode of cancer cell death induced by Re-diSe has still to be clarified, but the anticancer and immune effects of Re-diSe are demonstrated.
Conclusion
The efficacy of the Re-diSe was clearly demonstrated in this in vivo chicken embryo model with a significant reduction of the tumor weights at the low dose of 0.1 mg/Kg, which is safe. At the dose of 10 mg/kg, high inflammation and huge tumor necrosis were observed, with inflammatory infiltration of the CAM. Remarkably, no visible tumors were observed in more than 50% of the treated embryos at this dose. The immune effects suggested by the high inflammation score may be related to the known role of Re-diSe on the polarization of macrophages, via the decrease on cysteine proteases, and on the adaptive immunity through its indirect anti-oxidant effects.
Footnotes
Authors’ Contributions
Conceptualization: Philippe Collery, Adhikesavan Harikrishnan, Vijay Veena and Chloé Prunier; methodology, Chloé Prunier, Emilien Dosda and Jean Viallet; Synthesis of the Re-diSe drug: Didier Desmaële; validation: Chloé Prunier and Emilien Dosda; investigation: Chloé Prunier; resources: Philippe Collery; writing: Philippe Collery, Chloé Prunier and Didier Desmaële; project administration: Philippe Collery and Didier Desmaële; funding acquisition: Philippe Collery. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
Philippe Collery has been designated as inventor on a patent on “Rhenium complexes and their pharmaceutical use”. He is manager of the Society for the Coordination of Therapeutic Research, which is owner of the Intellectual Property Rights. The other Authors declare that they have no conflict of interest in relation to this study.
Funding
This research received no external funding.
- Received November 21, 2023.
- Revision received December 25, 2023.
- Accepted December 27, 2023.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
